African trypanosomes are medically important parasites and useful for the study of the organization of the mitochondrial genetic system, regulation of mitochondrial gene expression and mutant mitochondrial DNA alterations. I propose to detemine the genetic content of the two components of T. brucei mitochondrial DNA (K-DNA) and determine their physical and sequence relationships. Fundamental sequence structure, and sequence organization will be established and a restriction map of K-DNA will be constructed. The physical and sequence alterations of dyskinetoplastic mutant K-DNA will be determined. I further propose to characterize RNA transcribed from K-DNA and localize specific transcripts on the restriction map. I will determine if transcriptional control is the mechanism whereby expression of K-DNA is regulated. If this is the case, the transcription map will reveal the distribution of the regulated sequences. Whether dyskinetoplastic mutants are capable of K-DNA transcription will be ascertained. I will also simplify cultivation conditions for dyskinetoplastic mutants and screen for new K-DNA mutants. The results from these experiments will advance knowledge of kinetoplastid and other eukaryotic mitochondrial genetic systems and increase our understanding of eukaryotic sequence organizaton and regulation.